Epstein-Barr virus (EBV) is a human herpesvirus that infects over 90% of the world's population that persists as a latent infection in various lymphoid and epithelial malignancies, including Burkitt's lymphomas, nasopharyngeal carcinomas, gastric carcinomas, Hodgkin's and non-Hodgkin's lymphomas. The total number of EBV-associated malignancies is estimated to exceed 200,000 new cancers per year. The near universal presence of EBV in certain tumors suggests that new EBV-targeting therapies could be developed for these malignancies. Current chemotherapeutic treatments of EBV-positive cancers include broad-spectrum cytotoxic drugs that ignore the EBV positive status of tumors and have limited safety and selectivity. An alternative strategy, referred to as oncolytic therapy, utilizes drugs that stimulate reactivation of latent EBV to enhance the selective killing of EBV-positive tumors, especially in combination with existing inhibitors of herpesvirus lytic replicatio, like Ganciclovir (GCV). At present, no small molecule, including histone deacetylase (HDAC) inhibitors, have proven safe or effective in clinical trials for treatment of EBV-positive cancers.In an effort to develop new and more efficacious molecules for inducing EBV reactivation, we have developed high- throughput screening assays to identify a class of small molecules (referred to as the C60 series), that efficiently activate the EBV lytic cycle in multiple latency types, includng lymphoblastoid and nasopharyngeal carcinoma cell lines. This proposal aims to optimize this newly identified class of chemical probe using medicinal chemistry principles to enhance drug-like properties predictive of in vivo efficacy. We will also develop probes and methods to understand the biochemical mechanism of action of these EBV lytic activating agents. Finally, we will use EBV-associated cancer animal models to test the in vivo efficacy of advanced probes and lead compounds to reactivate the EBV lytic cycle more efficiently and with a significantly improved safety profile compared to HDAC inhibitors and phorbol esters. Central Hypothesis: A small molecule that induces lytic reactivation in EBV-positive tumors will provide a novel therapeutic approach to target EBV-positive malignant cells. The chemical probe will also provide new basic information on mechanisms controlling EBV latency.